A Mutation in the Surfactant Protein B Gene Responsible for Fatal Neonatal Respiratory Disease in Multiple Kindreds

A mutation in the surfactant protein B gene responsible for fatal neonatal respiratory disease in multiple kindreds.

L M Nogee, … , D E deMello, H R Colten

J Clin Invest. 1994;93(4):1860-1863. https://doi.org/10.1172/JCI117173.

Research Article

To determine the molecular defect accounting for the deficiency of pulmonary surfactant protein B (SP-B) in full-term neonates who died from respiratory failure associated with alveolar proteinosis, the sequence of the SP-B transcript in affected infants was ascertained. A frameshift mutation consisting of a substitution of GAA for C in codon 121 of the SP-B cDNA was identified. The three affected infants in the index family were homozygous for this mutation, which segregated in a fashion consistent with autosomal recessive inheritance of disease. The same mutation was found in two other unrelated infants who died from alveolar proteinosis, one of whom was also homozygous, and in the parents of an additional unrelated, affected infant, but was not observed in 50 control subjects. We conclude that this mutation is responsible for SP-B deficiency and neonatal alveolar proteinosis in multiple families and speculate that the disorder is more common than was recognized previously.

Find the latest version: https://jci.me/117173/pdf Rapid Publication

A Mutation in the Surfactant Protein B Gene Responsible for Fatal Neonatal Respiratory Disease in Multiple Kindreds Lawrence M. Nogee, * Gerard Gamier,t Harry C. Dietz, * Lori Singer,t Anne M. Murphy, * Daphne E. deMello,I and Harvey R. Coltent *Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; $Department of Pediatrics, Washington University School ofMedicine, St. Louis, Missouri 63110; and §Department ofPathology, St. Louis University School ofMedicine, St. Louis, Missouri 63117

Abstract (CAP) is a recognized familial cause of fatal respiratory disease in full-term infants (3-6). These observations suggest that an To determine the molecular defect accounting for the deficiency inherited deficiency of SP-B may cause this disorder and that of pulmonary surfactant protein B (SP-B) in full-term neonates abnormalities of surfactant proteins due to genetic mecha- who died from respiratory failure associated with alveolar pro- nisms could cause respiratory disease. To test these hypotheses teinosis, the sequence of the SP-B transcript in affected infants and to elucidate the molecular mechanisms responsible for the was ascertained. A frameshift mutation consisting of a substitu- lack of SP-B in these infants, we analyzed the SP-B transcripts tion of GAA for C in codon 121 of the SP-B cDNA was identi- in infants with CAP from unrelated families and compared fied. The three affected infants in the index family were homo- them with previously published SP-B cDNA and genomic se- zygous for this mutation, which segregated in a fashion consis- quences (7-10). tent with autosomal recessive inheritance of disease. The same mutation was found in two other unrelated infants who died Methods from alveolar proteinosis, one of whom was also homozygous, and in the parents of an additional unrelated, affected infant, Patients. The clinical course of infants from the index family (patients in We conclude that 11. 1, II.5, and II.6) and patient CAP-2 has been described previously (2, but was not observed 50 control subjects. 6). Patient CAP-3 was a 3-kg Caucasian female who developed respira- this mutation is responsible for SP-B deficiency and neonatal tory distress shortly after birth and who subsequently required intuba- alveolar proteinosis in multiple families and speculate that the tion, mechanical ventilation, and extracorporeal membrane oxygena- disorder is more common than was recognized previously. (J. tion. Open lung biopsy at 2 mo of age demonstrated characteristic Clin. Invest. 1994.93:1860-1863.) Key words: newborn * respi- findings of alveolar proteinosis: distal airspaces were filled with granu- ratory distress syndrome * pulmonary alveolar proteinosis lar, eosinophilic material that stained positively with periodic acid- bronchopulmonary dysplasia * lung diseases Schiff reagent, with foamy macrophages suspended in the protein- aceous material. She died at age 3 mo from respiratory failure; findings of alveolar proteinosis were also present at autopsy. Lung tissue from Introduction patients CAP-2 and CAP-3 was analyzed for SP-B content by immuno- histochemical staining and protein blotting as described previously A developmentally determined lack of pulmonary surfactant, (2). Control blood samples were obtained from healthy adult volun- the mixture of lipids and specific proteins that reduces alveolar teers without symptoms of pulmonary disease. surface tension, is recognized as the principal cause ofthe respi- cDNA preparation and sequencing. Total cellular RNA was pre- ratory distress syndrome in infants born prematurely (1). A pared from lung tissue frozen at the time of biopsy (patient 11.5) or deficiency of surfactant protein B (SP-B), ' an 8,000-D lung- rapid autopsy (patient II.6) by an acid phenol extraction method ( 11 ). specific protein, was recently demonstrated in three full-term Overlapping cDNA clones (spanning nucleotides 15-650, 22-761, siblings who died from respiratory failure associated with histo- 682-1405, and 1 172-1947) were generated by reverse transcription pathologic changes of alveolar proteinosis (2). SP-B has an (RT) of 5 jig of total cellular RNA using SP-B-specific antisense important role in the surface tension-lowering properties of primers, followed by PCR amplification, and subcloning the products surfactant, and congenital alveolar proteinosis into a plasmid vector (TAR cloning vector; Invitrogen, San Diego, CA) pulmonary according to the methods suggested by the manufacturer. For the most 5' and 3' sequences, genomic DNA sequences from nucleotides - 108 Address correspondence to Dr. Lawrence M. Nogee, Division of Neon- to 395 and 9052 to 9399 were amplified by PCR from patient II.6 and atology, CMSC 210, The Johns Hopkins Children's Center, 600 North subcloned. For the cDNA clone whose partial sequence is shown in Fig. Wolfe Street, Baltimore, MD 21287-3200. 1, the antisense SP-B-specific primer used was 5'-AGA GCC CTG Received for publication 18 October 1993 and in revised form 6 CAG AGC CAG CAA TAG GGG AGA-3', and the sense primer used December 1993. was 5'-ATG GCT GAG TCA CAC CTG CTG CAG TGG CTG-3'. 5 ,gg of plasmid DNA containing the cDNA insert was denatured and 1. Abbreviations used in this paper: CAP, congenital alveolar proteino- sequenced by the dideoxy chain termination method ( 12) (Sequenase, sis; RT, reverse transcription; SP, surfactant protein. V2.0; United States Biochemical Corp., Cleveland, OH) with incorpo- ration of 3S-dATP. The sequencing primer used was 5'-TTC CTG J. Clin. Invest. GAG CAG GAG TGC-3'. The products were separated on a 6% dena- © The American Society for Clinical Investigation, Inc. turing polyacrylamide gel and visualized by autoradiography. Se- 002 1-9738/94/04/1860/04 $2.00 quence analysis was repeated on two separately generated PCR prod- Volume 93, April 1994, 1860-1863 ucts from each of two affected siblings (patients II.5 and 11.6).

1860 Nogee, Garnier, Dietz, Singer, Murphy, deMello, and Colten A C G T T Figure 1. DNA se- A NORMAL SP-B cDNA G quence demonstrating _ l2lins2 mutation. The 1 2 3 4 5 6' 7 + 11 C DNA sequence 910 C ladder 2Q0 Kb c of the sense strand of a A * cDNA B SP-B DEFICIENT cDNAs A clone from the G * index infant with SP-B C-GAA C A ; deficiency (patient 11.5) 1 2 4 5 6 *8 11 T 1 AC910 AAAA C in the region of the mu- A tation is shown. The 375 967 1316 1972 T I.3'8 normal SP-B cDNA se- C- GAA GC C 119 120 121 122 123 quenceislistedbelow -- ACA for comparison. In co- 1 2 |3 4 5 6 7 8 910 11 A Tyr Phe Pro Leu Val * * - AAA NORMALTOTTC ~CCTG GTC . NORMAL TAC TTC PCC CTG GTC donsubstitution121 thereof isGAAa 3-bpfor 375 696.7 967 316 1972 4316 SP-B DEFICIENT: TAC TTC M CCC TGG C, marked with aster- Tyr Phe Asp Pro Trp isks. Figure 2. Aberrant splicing of SP-B-deficient transcripts. The normal SP-B mRNA structure is depicted in A, with the relative size of each exon indicated. The narrow bars indicate the untranslated regions. The two SP-B-deficient transcripts are depicted in B. The position of Genomic DNA preparation and restriction analysis. Genomic DNA the 12 1ins2 mutation in exon 4 is shown. At where the was prepared from whole blood positions lymphocytes (patients 1.1, I.2, 11.2, published cDNA sequences differ (7-10), representing apparent po- II.3, 11.4, and controls), frozen lung tissue (patients II.5, II.6, CAP-2, lymorphisms, the nucleotide sequence of the deficient infants is and CAP-3), or formalin-fixed paraffin-embedded lung tissue (patient shown. Each transcript was represented at least two different II. 1) as described by previously (13, 14). 200 ng of genomic DNA was cDNA clones generated by RT-PCR from II.5 or *Pre- used in the PCR. After patients II.6. denaturation at 950C for 5 min, 35 cycles of mature termination codon. These sequence data are available from denaturation at 950C for 30 s, annealing at 590C for 30 s, and extension GenBank under accession numbers M2446 1, M 16764, J0276 1, and at 720C for 45 s were performed using a thermal cycler (National Lab- M19097. net Co., Woodbridge, NJ) followed by a 10-min incubation at 720C. The primers used were 5'-GGC CTT GTG TCC AGG GAC-3' in the sense orientation and 5'-TGT GTG TGA GAG TGA GGG TGT the 3' untranslated region and reported variants at positions AAG-3' in the antisense orientation ( 1 5), corresponding to positions 696, 697, 967, 1318, and 1972 1383-1400 and 2135-2158, (7-10). respectively, of the SP-B gene sequence The 121ins2 mutation introduces new restriction sites (7), which are predicted to for amplify a 776-bp DNA fragment. After SfuI and To determine whether amplification, a l6-,d aliquot of the PCR reaction was digested with 15 (TT/CGAA) TaqI (T/CGA). U of SfuI (Boehringer Mannheim affected infants carried this mutation on both alleles, a 776-bp Corp., Indianapolis, IN) using re- DNA agents and conditions supplied by the manufacturer. The resulting fragment spanning the restriction site was amplified products were separated on a 2.0% agarose gel and visualized by ethi- from genomic DNA from each of the three infants with SP-B dium bromide staining. deficiency in this family. Treatment of the amplified products with SfuI completely digested the 776-bp fragment to 61 1 and Results 165 bp in each of the affected infants, indicating that they were homozygous for this mutation. SfuI digestion of PCR-ampli- SP-B cDNA was detectable by RT-PCR in the lung tissue ofthe fied genomic DNA from both parents indicated that both were index patient with SP-B deficiency and alveolar proteinosis, heterozygous for the 12 1ins2 mutation. Ofthe three living chil- although the amount of SP-B-deficient cDNA was markedly dren, who along with their parents have no symptoms of pul- less than that generated by RT-PCR from RNA obtained from monary disease, two are heterozygous for the mutation, and control lung tissue (not shown). Sequence analysis of the SP- one is homozygous for the normal allele (Fig. 3). B-deficient cDNA clones revealed a substitution of three bases (GAA) for the single nucleotide (C) at position 375 ofthe SP-B cDNA, disrupting codon 121 (Fig. 1 ) (referred to as 121 ins2). 4 -- Figure 3. Restriction This sequence was found in all cDNAs generated from each set 1 2 analysis for the 12 lins2 of primer pairs that spanned this region. Direct sequence analy- --- mutation in a family 11 sis of genomic DNA from this infant and an affected sibling % I. with SP-B deficiency. (patient 11.6) confirmed this sequence. The net gain of two 1 2 33 4 5 6 The patterns after SfuI _ digestion of a bases causes a frameshift, introducing a premature signal for 776-bp - 776 DNA termination of translation after codon 214. bp fragment contain- - 611 bp ing the site of the Analysis of SP-B-deficient cDNA clones from both the in- l2lins2 mutation 165 dex patient and an affected sibling (patient II.6) for the se- bp from the 5' end are quence distal to the 121 ins2 mutation indicated the presence of shown below the corre- two transcripts, present in approximately equal amounts as - 165bp sponding symbol for determined from multiple PCR reactions. In one of these tran- each family member. scripts exon 7 was deleted, and in a second transcript exon 7 Lines denote bands at was present, but a cryptic splice site 12 bp 3' of 776 (undigested) and the reported 61 1 and 165 Affected exon 8 splice junction was used (Fig. 2). The SP-B bp (digested). infants (II.l, II.5, and II.6) are cDNA did homozygous for the restriction site; both parents (I. 1 and 1.2) and not differ otherwise from the published SP-B DNA sequences, two of their living children (II.2 and II.4) are heterozygous, and a except for a previously unreported single base substitution in third child (II.3) is homozygous normal.

Surfactant Protein B Gene Mutation 1861 applied to infants with CAP from other families. In this report, we demonstrate a recurrent mutation in the SP-B gene account- 0 G. CONTROLS ing for the lack of SP-B protein in multiple kindreds, establish- ing the lack of SP-B as the primary basis for disease in these families and SP-B deficiency as a common cause of CAP. 1353 bp Whether inherited abnormalities of other surfactant proteins 1078 bp produce similar disease is unknown. While the mechanism(s) 872 bp whereby SP-B deficiency leads to the histopathological appear- 603 bp ance of alveolar proteinosis remains unclear, these findings imply a role for SP-B in the metabolism of SP-A, SP-C, and 310 bp surfactant phospholipids. The frameshift and consequent premature termination co- -_- 194 bp don clearly account for the lack of the 8,000-D SP-B protein, 118 bp which is derived from codons 201 to 279 of the 381-amino acid proprotein, and likely account for the marked reduction in SP-B mRNA. The introduction of nonsense codons has been Figure 4. Demonstration of 12 lins2 mutation in two unrelated in- associated with a marked reduction in transcript level from the fants with SP-B deficiency and alveolar proteinosis. SfuI restriction mutant allele of other genes including f3-globin ( 17 ), aI-anti- digests of a 776-bp DNA fragment containing the site of the 121 ins2 trypsin deficiency ( 18), cystic fibrosis transmembrane regula- mutation 165 bp from the 5' end are shown for two infants with al- tor (19), fibrillin (20), and the insulin receptor (21). The veolar proteinosis and SP-B deficiency (left panel) and for 3 of 50 mechanism(s) leading to reduction in mRNA may be related control patients without lung disease (right panel). The sizes of mo- to alterations in RNA processing (22-24). The mechanisms lecular weight standards (far left and far right) are shown on the right. and significance of the abnormalities in SP-B RNA splicing Patient CAP-2 was homozygous for the restriction site, and patient 2) are unclear. Both alter- CAP-3 had this mutation on one allele. DNA from control subjects observed in the index patients (Fig. was not digested. ations are distal to the premature termination codon, which is in exon 6, and are thus unlikely to be causally related to the deficiency of SP-B protein. Abnormalities of RNA splicing To ascertain whether the 121 ins2 mutation was unique to have been observed in mutant alleles of other genes containing this family, the presence of this mutation was looked for in the premature termination codons (20, 25). Alternatively, these DNA of two other unrelated, full-term infants who died from splicing abnormalities may also occur in normal tissue and respiratory failure associated with alveolar proteinosis and ab- were selected because of the marked reduction in normal SP-B sent SP-B protein in their lung tissue. Restriction analysis dem- transcript levels. onstrated that one of these two infants was homozygous for the The incidence of SP-B deficiency associated with alveolar 121 ins2 mutation, and the other had the 121 ins2 mutation on proteinosis is unknown. Affected infants also have features of only one allele, suggesting that she was a compound heterozy- respiratory distress syndrome, persistent pulmonary hyperten- gote for two different SP-B gene defects (Fig. 4). Additionally, sion, and bronchopulmonary dysplasia and may not be recog- the parents of another infant with CAP (5), from whom tissue nized without autopsy or biopsy data or until a second child in was not available, were both found to carry this mutation on the same family is affected (2, 6). It is therefore likely that the one allele (not shown), implying that their affected child was syndrome has been underreported. Finding the identical muta- homozygous. In contrast, restriction analysis ofPCR-amplified tion on seven of eight chromosomes from unrelated infants genomic DNA obtained from 50 unrelated adult controls with- raises the possibility that the disease may be more common out evidence of lung disease did not show the presence of this than has been appreciated and indicates that molecular tech- restriction site on any of the 100 chromosomes analyzed. In 11 niques will be useful in diagnostic testing. Whether the pres- ofthe control patients, two bands of equal intensity were gener- ence of the same mutation in unrelated individuals is because ated by PCR (not shown). This region of the SP-B gene is of a mutation arising in a common ancestor or reflects a mecha- known to be polymorphic, containing dinucleotide repeats of nism causing a recurrent de novo mutation at this site is un- variable length ( 15). known. The finding of an apparent compound heterozygote SP-B-deficient patient suggests there is some molecular hetero- Discussion geneity of this disorder. At the present time there is no effective treatment for neona- CAP is a uniformly fatal lung disease characterized clinically tal alveolar proteinosis, and gene therapy may be required to by respiratory failure in the neonatal period and histopathologi- treat this disorder. Gene therapy trials for SP-B deficiency, cally by the accumulation of granular, eosinophilic, periodic which has a rapidly progressive course, may be helpful in devel- acid-Schiff reagent-positive staining material in the distal air- oping strategies for treatment of other genetic illnesses such as spaces. The recent observation that three siblings with this dis- cystic fibrosis and a1-antitrypsin deficiency which lead to more order lacked SP-B protein suggested that an inherited defi- gradually progressive pulmonary disease. ciency of SP-B was the cause of this disease. However, abnormalities of other surfactant components, including increased immunostaining for SP-C, an altered distribution of SP-A Acknowledgments staining, and a markedly abnormal amniotic fluid phospho- lipid profile, were also observed in the SP-B-deficient infants We thank Drs. Joel Haas, T. Allen Memitt, Henri Krous, and Paul (2, 16). Additionally, it was unknown if those observations Dykema for providing patient specimens; Dr. Garry R. Cutting for

1862 Nogee, Garnier, Dietz, Singer, Murphy, deMello, and Colten providing control DNA specimens; and Larry Jones, Ling Peng, and 12. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with Ray Kendzior, Jr. for technical assistance. chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74:5463-5467. 13. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A This work was supported in part by grants from the National Insti- Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. tutes of Health (HL-37591 to H. R. Colten, HL-34748 to D. E. de- 9.17-9.19. Mello, HL-50184 to A. M. Murphy, and HL-02815 to H. C. Dietz). L. 14. Rogers, B. B., L. C. Alpert, E. A. S. Hine, and G. J. Buffone. 1990. Analysis M. Nogee is the recipient of a Basil O'Connor Scholar Award from the of DNA in fresh and frozen tissue by the polymerase chain reaction. Am. J. March of Dimes, and A. M. Murphy is the recipient of a Clinician Pathol. 136:541-548. Scientist Award from the American Heart Association. 15. Todd, S., and S. L. Naylor. 1991. Dinucleotide repeat polymorphism in the human surfactant-associated protein 3 gene (SFTP3). Nucleic Acids Res. 19:3756. References 16. Nogee, L., W. Hull, J. Whitsett, A. Hamvas, and F. S. Cole. 1993. In- herited surfactant protein B deficiency: prenatal diagnosis. Pediatr. Res. 33:227a. (Abstr.) 1. Farrell, P. M., and M. A. Avery. 1975. Hyaline membrane disease. Am. 17. Baserga, S., and E. J. Benz, Jr. 1988. Nonsense mutations in the human Rev. Respir. Dis. 111:657-688. f3-globin gene affect mRNA metabolism. Proc. Natl. Acad. Sci. USA. 85:2056- 2. Nogee, L. M., D. E. deMello, L. P. Dehner, and H. R. Colten. 1993. Defi- 2060. ciency of pulmonary surfactant protein B in congenital alveolar proteinosis. N. 18. Satoh, K., T. Nukiwa, M. Brantly, R. I. Garver, Jr., M. Hofker, M. Engl. J. Med. 328:406-4 10. Courtney, and R. G. Crystal. 1988. Emphysema associated with complete ab- 3. Coleman, M., L. P. Dehner, R. K. Sibley, B. A. Burke, P. R. L'Heureux, sence of a I -antitrypsin of a stop codon in an a I -antitrypsin-coding exon. Am. J. and T. R. Thompson. 1980. Pulmonary alveolar proteinosis: an uncommon Hum. Genet. 42:77-83. cause of chronic neonatal respiratory failure. Am. Rev. Respir. Dis. 121:583-586. 19. Hamosh, A., B. C. Trapnell, P. L. Zeitlen, C. Montrose-Rafizadeh, B. J. 4. Knight, D. P., and J. A. Knight. 1985. Pulmonary alveolar proteinosis in Rosenstein, R. G. Crystal, and G. R. Cutting. 1991. Severe deficiency of cystic the newborn. Arch. Pathol. Lab. Med. 109:529-531. fibrosis transmembrane conductance regulator messenger RNA carrying non- 5. Schumacher, R. E., A. J. Marrogi, and K. P. Heidelberger. 1989. Pulmo- sense mutations R553X and W1316X in respiratory epithelial cells of patients nary alveolar proteinosis in a newborn. Pediatric Pulmonology. 7:178-182. with cystic fibrosis. J. Clin. Invest. 88:1880-1885. 6. Moulton, S. L., H. F. Krous, T. A. Merritt, R. M. Odell, E. Gangitano, and 20. Dietz, H. C., D. Valle, C. A. Francomano, R. J. Kendzior, Jr., R. E. J. D. Cornish. 1992. Congenital alveolar proteinosis: failure of treatment with Pyeritz, and G. R. Cutting. 1993. The skipping of constitutive exons in vivo extracorporeal life support. J. Pediatr. 120:297-302. induced by nonsense mutations. Science (Wash. DC). 259:680-683. 7. Pilot-Matias, T. J., S. E. Kister, J. L. Fox, K. Kropp, S. W. Glasser, and J. A. 21. Kadowaki, T., H. Kadowaki, M. M. Rechler, M. Serrano-Rios, J. Roth, P. Whitsett. 1989. Structure and organization of the gene encoding human pulmo- Gorden, and S. I. Taylor. 1990. Five mutant alleles ofthe insulin receptor gene in nary surfactant proteolipid SP-B. DNA (NY). 8:75-86. patients with genetic forms of insulin resistance. J. Clin. Invest. 86:254-264. 8. Glasser, S. W., T. R. Korfhagen, T. Weaver, T. Pilot-Matias, J. L. Fox, and 22. Urlaub, G., P. J. Mitchell, C. J. Ciudad, and L. A. Chasin. 1989. Nonsense J. A. Whitsett. 1987. cDNA and deduced amino acid sequence of human pulmo- mutations in the dihydrofolate reductase gene affect RNA processing. Mol. Cell. nary surfactant-associated proteolipid SPL(Phe). Proc. Nat!. Acad. Sci. USA. Biol. 9:2868-2880. 84:4007-4011. 23. Belgrader, P., J. Cheng, and L. E. Maquat. 1993. Evidence to implicate 9. Jacobs, K. A., D. S. Phelps, R. Steinbrink, J. Fisch, R. Kriz, L. Mitsock, J. P. translation by ribosomes in the mechanism by which nonsense codons reduce the Doughterty, H. W. Taeusch, and J. Floros. 1987. Isolation of a cDNA clone nuclear level of human triosephosphate isomerase mRNA. Proc. Natl. Acad. Sci. encoding a high molecular weight precursor to a 6-kda pulmonary surfactant-as- USA. 90:482-486. sociated protein. J. BioL. Chem. 262:9808-981 1. 24. Cheng, J., and L. E. Maquat. 1993. Nonsense codons can reduce the 10. Revak, S. D., T. A. Merritt, E. Degryse, L. Stefani, M. Courtney, M. abundance of nuclear mRNA without affecting the abundance of pre-mRNA or Hallman, and C. G. Cochrane. 1988. Use of human surfactant low molecular the half-life of cytoplasmic mRNA. Mol. Cell. Biol. 13:1892-1902. weight apoproteins in the reconstitution of surfactant biologic activity. J. Clin. 25. Fisher, C. W., C. R. Fisher, J. L. Chuang, K. S. Lau, D. T. Chuang, and Invest. 81:826-833. R. P. Cox. 1993. Occurrence of a 2-bp (AT) deletion allele and a nonsense (G-to- 11. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isola- T) mutant allele at the E2 (DBT) locus of six patients with maple syrup urine tion by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Bio- disease: multiple-exon skipping as a secondary effect of the mutations. Am. J. chem. 162:156-159. Hum. Genet. 52:414-424.

Surfactant Protein B Gene Mutation 1863